JPH0647541B2 - Method for producing novel hemin complex compound for medical use - Google Patents

Description

The present invention relates to a process for the preparation of physiologically active water-soluble hemin alginate or hemin lysinate compounds, which compounds are used as tablets or capsules or are, for example, sterile saline solution. It is intended for use as a dry substance for injection which can be reconstituted with.

Hemin is present in organisms as a prosthetic group of hemoglobin, and is present in most cytochromes and certain enzymes. Hemoglobin is synthesized in bone marrow. Although hemin is released when hemin protein is decomposed, under normal physiological conditions, only a small part of the released hemin is used for the synthesis of new hemin protein. Hemin is decomposed into biliverdin by the action of hemin oxygenase, and biliverdin is further reduced to bilirubin. Naturally, intact natural hemoglobin does not serve as a substrate for hemin oxygenase.

The defect in hemoglobin synthesis can be said to be due to inhibition of hemin or globin chain synthesis. Hemin synthesis is a) deficiency of certain components required for synthesis, or b)
It can be said that the enzyme that catalyzes the synthesis is disturbed by the functional disorder.

a) Iron deficiency is a limiting factor in hemin synthesis. Organisms consume the required daily amount of iron (1-2 mg) from their diet. Iron deficiency can result from eating a diet that is deficient in iron and, in some cases, from the presence of iron-binding compounds in the diet. Moreover, even if iron is present in an appropriate amount in the diet, if iron absorption mechanism is disturbed, iron deficiency may similarly occur. For whatever reason, iron deficiency leads sooner or later to anemia.

By chance, vitamin B ₆ deficiency interferes with iron utilization by cells, even though iron absorption is normal. as a result,
Some kind of sideroblast anemia appears.

Treatment of anemia due to iron deficiency involves the oral administration of iron drugs (eg,
Ferrous sulphate or ferrous gluconate) or, rarely, by injection (iron sorbitol). If the iron absorption mechanism is disturbed, these conventional oral agents become useless; that is, iron cannot penetrate even into the intestinal mucosal cells. In contrast to inorganic iron, hemin iron, where iron is bound to hemin, is found in these intestinal mucosal cells even if the iron absorption mechanism is disturbed and normal oral treatment is not possible. It will be absorbed. Therefore, hemin iron is the only known effective oral therapeutic drug when oral treatment is not possible. It is known that hemin iron is absorbed 4-5 times better than inorganic iron, even in totally healthy subjects (Sepnen H and Tak).
kunen H: Suomen Lkrileh
ti36: 2071-2072,1981).

b) Hemin synthesis is enzymatically regulated. The dysfunction of the enzyme that catalyzes hemin synthesis can be carried out genetically or by an external factor. When the function of the enzyme is impaired, it leads to an invariably reduced production of hemin, which is manifested in the development of porphyria, some sideroblast anemia or other diseases.

Porphyrias belong to the most important group of diseases caused by the impaired enzymatic function. In patients with porphyria, porphyrin, an intermediate product of hemin synthesis, is deposited, and the secretion of porphyrin into urine and excretion is increased. Most porphyrias manifest themselves as acute seizures, which are extremely difficult to overcome.

Occasionally, dysfunction of enzymes involved in hemin synthesis results in the development of different types of sideroblast anemia, instead of manifesting porphyria. Sideroblast anemia is also congenital and acquired.

To date, the treatment of porphyria has been based primarily on the evasion of certain drugs and the massive administration of carbon hydrates during acute attacks, but the effect is modest. It was a thing. Since the etiology of porphyria has been clarified, the treatment by intravenous administration of hemin compound (hematin) has been continuously advanced. Although hematin has been shown to be effective in treating stroke attacks of porphyria, it has become the etiology of thrombophlebitis in more than 50% of patients. Furthermore, since hematin is unstable, it lacks stability for producing it on an industrial scale. Therefore, the potential for effective treatment of patients with porphyria is very low.

The object of the present invention is to provide a water-soluble hemin iron compound.
This compound is for treating some types of anemia by the iron in the hemin molecule, so to speak. The compound is initially intended for the treatment of porphyrias where the normal production of hemoglobin is prevented for some or other reasons. In addition to injection, this compound is intended for oral administration as tablets or capsules, so it must be water-soluble.

Hemin is only slightly soluble in water and can be obtained in pure form from blood by extracting an aqueous solution of hemolyzed red blood cells with a mixture with hydrochloric acid or acetic acid. Also known is a method based on the extraction of hemin with acetone at pH 7.0, for example in the presence of histidyl histidine, pilocarpine or imidazole (Wakid
NW and Helou KY: Int. J. Biochem. 4 : 259-2
67, 1973).

PCT Patent Application No. 813749 (PCT / FI81 /
[00026) discloses a method for preparing a water-soluble hemin concentrate. In this water-soluble hemin concentrate, about 40 W / W% of which is hemin, and the remaining part is a "blood substance of unknown property". (Blood substance) ". This product is intended to be used in the form of a lyophilisate, as an iron supplement in foods or as an anti-anemic agent.

The disadvantage of this method is that the end product is hemin and "blood substances".
It is a mixture with. This mixture is not suitable for injection due to the inhomogeneity of this latter component.

In hospitals, porphyrias have been treated with a mixture of hemin dissolved in sterile sodium carbonate solution (hematin) at the time of use and treated with this mixture. Since this solution is unstable, it cannot be industrially manufactured on a large scale as a commercial product. Moreover, in about 50% of cases, hematin administration causes thrombophlebitis at the injection site, presumably due to the high pH of the solution. This is a serious drawback that considerably reduces the usefulness of the product.

The present invention relates to a method for preparing a water-soluble hemin compound, which method is suitable, for example, L-lysine or L-arginine and a suitable amino acid, for example in a solvent mixture containing water and acetone. It is carried out by reacting a base with a low-water-soluble crystallized hemin. It is quite surprising that the composition of the solvent, ie the ratio of organic solvent to water, is very important for increasing the medical value of the final product. Due to the very low water content in the solvent mixture (about 7%), neither hemin nor L-arginine, which normally dissolves fairly easily, will also dissolve. The reaction is caused by vigorous stirring, and the pH of the solution is always controlled. The product formed is separated and dried; the hemin compound thus obtained is in a dried form and is water-soluble, which is not only a point of view of pharmaceutical technology but also of a medicine. It is also an important point from the aspect.

The hemin molecule contains two carboxyl groups, which react with the basic amino group of L-lysine or L-arginine.

The hemin alginate produced according to the present invention.
ginate) and hemin lysinate were dissolved in water and the solution was taken, and also hemin and L
-Measuring a mixture of arginine and water dissolved in water, the pH of each is measured at different times,
The two were compared. The results are shown in Table 1. From the results of pH measurement, the pH of hemin alginate and hemin lysinate is stable for up to 24 hours (pH about 8)
I understand. On the other hand, the pH of the mechanically mixed one is very slow and lower, probably because the reaction between the carboxyl of hemin and the amino group of L-amino acid is extremely slow. Seem. Therefore, this method does not yield a therapeutically useful product. The hemin alginate and hemin lysinate prepared according to the present invention are complex compounds (co-compound) formed by reacting an L-amino acid with a carboxyl of hemin.
mplex compound).

On the one hand, to determine the optimum molar ratio between these two reactants, and on the other hand, to determine the optimum composition of the solvent mixture, the following tests were carried out on hemin and arginine: organic solvent and water. Crystalline hemin and L-arginine were reacted at a molar ratio of 1: 2 and 1: 3 in a solvent mixture in which the mixing ratio of (1) was changed with vigorous stirring. The precipitate formed was removed, washed and dried.

Approximately 1.0 g of hemin alginate obtained in each test
Was dissolved in 50 ml of distilled water with vigorous stirring for about 1 hour, and the solubility in water was measured.

The solution is centrifuged (about 3500 rpm) and the residue is distilled water 1
Rinse with 0 ml and 10 ml of acetone, then dry,
The weight was measured. The insoluble residue consists mainly of unreacted hemin. The test results are shown in Table 2.

A tar-like material was formed in some of the tests described above, but this tar-like material could not be powdered.

It has been found that a practically suitable molar ratio of hemin to arginine is 1: 3. It has been found that the optimum solvent mixture is a mixture of 300 ml of acetone and 20 ml of water. The reason is that hemin requires a slight excess of L-arginine over the exact reaction amount.

The hemin compound was added to the external ear vein (auricu) of california white rabbits.
larveim) at 5 mg / kg to study the local effect of surrounding tissue by intravenous infusion of hemin compounds. As a reference solution, normal hemin carbon dioxide
ate) solution (hematin) was used.

The tissue surrounding the vein remained normal after infusion of the hemin alginate solution. That is, aseptic inflammation (steril
e inflammation) (thrombus phlebitis) has never occurred.

Similar results were seen after injection of the corresponding hemin lysinate solution. It can therefore be concluded that these hemin compounds do not cause thrombophlebitis when they are infused intravenously.

When hemin carbonate solution was similarly administered, the tissue surrounding the vein became red and hypersensitivity; that is, a clear aseptic inflammation (thrombophlebitis) developed. Thrombophlebitis still developed 3 days after the injection of the hemin carbonate solution.

The physiological properties of various water-soluble hemin compounds were evaluated by testing the ability of the hemin oxygenase to degrade hemin compounds. Methhemalbumin, a physiological substrate for hemin oxygenase, is broken down by this enzyme into biliverdin, which is further reduced to bilirubin by biliverdin reductase. Therefore, first of all, excess hemin, which is not available to the organism, is broken down by hemin oxygenase into bilirubin and other closely related substances, which are normally excreted. Therefore, the reaction rate limiting enzyme is hemin oxygenase.

We first performed an enzymatic assay to discover the ability of hemin alginate and hemin lysinate to act as substrates for hemin oxygenase, to give an example, in which the activity of the reference substrate, methhemalbumin, was determined. Will be expressed as 100. The corresponding value obtained above for hemin alginate and hemin lysinate was 106. The activity of other amine derivatives of hemin, ie, where the amine component is diethanolamine, ethylamine, cyclohexylamine or piperidine, is 13, respectively.
It was 21, 31 and 78. These tests show that hemin alginate and hemin lysinate
For hemin oxygenase, it shows that it behaves in organisms in the same manner as normal physiological compounds.

The invention is best illustrated by the following examples.

Example 1 equipped with a mechanical stirrer, 300 ml of acetone and 20 ml of water
In a beaker containing a solvent mixture consisting of crystalline hemin 6.
52 g (0.01 M) and crystalline L-arginine 3.48 g (0.02
M) was added and vigorously stirred for 10 to 15 hours. The product formed was removed, washed with acetone and dried.

Yield of hemin alginate: 9.5 g (95%). Insoluble residue determined by the method described above: 0.14 g (14
%).

Example 2 6.52 g (0.01 M) crystalline hemin and 4.36 g (0.025 M) crystalline L-arginine were treated as described in Example 1.

Yield of hemin alginate: 11.9 g (about 100%). Insoluble residue: 0.042 g (4.2%).

Example 3 6.52 g (0.01 M) crystalline hemin and 5. L-arginine crystalline.
23 g (0.03M) was treated as described in Example 1.

Yield of hemin alginate: 12.0 g (about 102%). Insoluble residue: 0.001 g (0.1%).

Example 4 6.52 g (0.01 M) crystalline hemin and 6. L-arginine crystalline.
10 g (0.035M) was treated as described in Example 1.

Yield of hemin alginate: 12.0 g (95%). Insoluble residue: 0.0005 g (0.05%).

Example 5 6.52 g (0.01 M) crystalline hemin and 4.39 g crystalline L-lysine
(0.03M) was treated as described in Example 1.

Yield of hemin lysinate: 10.8 g (99%). Insoluble residue: 0.020 g (2.8%).

The optimal molar ratio of hemin to alginate appears to be 1: 3 (Example 3). The reason is that at this molar ratio, the yield of hemin alginate is the highest, while the amount of insoluble residue is the lowest.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Rhino Orabi Tokora Finland, 00260 Helsinki 26 Mannell Haiminte 56 E 42 (72) Inventor Rimo Antero Tenhaunen Finnland, 00100 Helsinki 10 Beinemeisen Katsu 1A (56) Reference JP-A-55-9023 (JP, A) JP-A-57-80317 (JP, A) JP-A 57-209211 (JP, A) JP-A-49-30521 (JP, A)

Claims (4)

[Claims] 1. A novel, physiologically active, water-soluble water for use as a raw material for tablets or capsules or as a dry substance for preparing an injection, for the treatment of various anemias, particularly anemia associated with porphyria. A method for preparing a hemin compound by reacting hemin with a basic amino acid, wherein the basic amino acid is L-arginine or L-lysine, and the molar ratio of hemin to the amino acid is 1: 1 to 1: 4. And reacting both in a mixture of an organic solvent and water with vigorous stirring at room temperature for 10 to 15 hours to form a complex compound consisting of hemin alginate or hemin lysinate, respectively. 2. The method according to claim 1, wherein the solvent mixture is composed of acetone and water, and the mixing ratio thereof is 300: 10 to 300: 25 V / V. 3. A method according to claim 2, characterized in that the ratio is 300: 20. 4. A water-soluble hemin alginate or hemin lysinate produced by the reaction of hemin with L-arginine or L-lysine for treating various anemias, particularly anemia associated with porphyria. , In powder form in capsules, or as a dry substance for injection after reduction with sterile saline solution, or in tablets together with a carrier. The method according to claim 1.